Discussion
While COVID-19 disease presents primarily with respiratory symptoms, for
many patients including children, it is a systemic disease with a wide
range of effects on many organs (39-41). In this report, we show
preliminary evidence for association of caspase molecules that play role
in cell death and immunity, not only in the acute phase but also late
stages of COVID19 (42). The changes are seen in multiple caspase
molecules, and a number of different circulating blood cells, a finding
that will further lead to exploration of the systemic nature of this
disease.
Caspase-1 has been proposed to play role in the pathophysiology of
COVID-19 (43). In addition to leading to a lytic form of cell death
called pyroptosis, caspase-1 induces the formation of biologically
active IL-18 and IL-1b (26, 44). IL-18 induces an IFN-γ response, while
IL-1β induces neutrophil influx and activation, T and B-cell activation,
cytokine and antibody production, and promotes Th17 differentiation
(45-48). Elevated levels of IL-18, IL-1β, and other proinflammatory
cytokines were observed from the lungs and sera of COVID-19 patients
(49). Although activation of the inflammasome enhances immunity against
pathogens, the accompanying danger and inflammatory signals originating
from pyroptosing immune system cells (e.g., T cell and
macrophage/dendritic cells) can be damaging to the host in several ways.
First, it will result in immune cell lymphopenia, such as that observed
with T cells, a pathognomonic feature for SARS-CoV-2, creating an
adaptive immune defect. Second, the host will have difficulty
controlling the inflammation created in the setting of this immune
deficiency as the “danger signals” would also be originating from
dying immune system cells (50-52). The end result is likely a
self-damaging shut down of the immune system, resulting in acute
virus-induced immune deficiency (AVID). Preventing the pyroptotic
lymphocyte death by using caspase inhibitors may lead to better success
rather than inhibition of the inflammatory response from the cell death
itself. The failure of cytokine targeted therapies could be due to that
adaptive immune dysfunction due to AVID weighing more heavily than an
inflammatory response in disease progression (18).
In our active T-cell caspase-1 assay, we analyzed the sensitivity to
nigericin stimulation. This provided further information on the cell
surface pannexin-1 expression, which is upregulated by cellular caspases
and can play role in disease pathogenesis. Also, cell surface expression
level of pannexin-1 can vary between healthy controls, which can explain
the differences in response to nigericin. We also found that EMR is
effective in reducing active caspase-1 T cells from COVID-19 patients,
while VX765 failed to significantly do so. VX765 is a prodrug that needs
hydrolyzation to form into its active form and is a reversible inhibitor
of caspase-1, as opposed to EMR which is an irreversible inhibitor.
Furthermore, EMR is transported into cells via active transport with
cell membrane channels, whereas VX765 is internalized by passive
diffusion. All these factors may explain the differences we see between
these two caspase inhibitors. Further studies are needed to explain the
differences in inhibition between the two molecules, particularly in the
context of SARS-CoV-2 infection.
The changes in caspase expression are not only limited to T cells, as we
show changes in caspase-3 in RBCs and caspase-5 in neutrophils. Caspase
activation has been shown to induce changes in the RBC morphology
(53-55), which can explain the contamination of the PBMC layer during
cell separation as a result of a reduction in their density.
Furthermore, their overexpression of caspase 3/7 can subsequently
contribute to the formation or advancement of inflammatory microvascular
thrombi, which is prominently found in the lung, kidney, and heart of
patients with COVID-19 (56, 57). Although viral illnesses typically will
impact the function or the life-cycle of lymphocytes, presenting with
either lymphocytosis, such as in with CMV, influenza, varicella, or more
rarely, lymphopenia, as in H5N1, H1N1, HIV, the finding of neutrophilia
in the setting of moderate to severe COVID-19 has been a common, but
intriguing finding (58). In the absence of significant overexpression of
apoptotic caspases, the increase in the inflammatory caspase-5 in
neutrophils may play a part in the neutrophilia observed with COVID-19.
Furthermore, the production of IL-10 by neutrophils with increase
caspase activity, can further suppress the proliferation of T
lymphocytes, hence contributing to the adaptive immune deficiency.
Caspase molecules have been studied extensively in many forms of
inflammatory conditions, such as obesity, diabetes and nonalcoholic
steatohepatisis (NASH) (59-61). Caspase-1-dependent inflammasome
activation has been shown to have a crucial function in the
establishment of diabetic nephropathy (62). In an animal model of
hypertension apoptosis of myocardial cells were demonstrated, and the
apoptosis becomes more serious with the constantly elevated level and
prolonged duration of hypertension. The activity of caspase‑3 was shown
to have a close correlation with cardiomyocyte apoptosis (63). Our data
showing increased expression of active caspase-1 in T-helper cells of
patients with asthma and immune deficiencies correlates with their
high-risk classification for severe COVID-19 as provided by the centers
for disease control (CDC). Perhaps, the changes in cellular caspases
seen in COVID-19 may not only explain the multisystem involvement in
this disease but may allow for identification for those at risk for
complications, including long haulers, based on caspase expression in
blood cells.
Our findings suggest a novel alternate therapeutic approach against
COVID-19 through the use of caspase inhibition early on in the course of
infection to alleviate or prevent disease progression. As an oral
formulation, EMR has been shown to reduce serum markers of apoptosis
(caspase-3/7), liver enzymes, function (e.g., reducing ALT, MELD &
Child-Pugh scores, INR and total bilirubin) and inflammatory biomarkers
(CK-18) in patients w/ hepatitis C virus and NASH (64). Although there
was no improvement in liver histology, it is possible that the pathology
of this disease has mechanisms that are caspase-independent or with the
timing of therapy (65, 66). Although SARS-CoV-2 does not seem to infect
immune system cells (with the possible exception of macrophage or
dendritic cells), the outcome of T cell depletion in severe forms of the
disease seems to be through caspase-1 activation, a mechanism also
proposed in HIV (67). A better understanding of the impact of different
co-morbid conditions on T cell caspase expression at baseline, before
exposure to SARS-CoV-2, may identify those that are at highest risk for
developing severe disease. There is a large body of evidence pointing
out to an activated inflammasome in a wide variety of disorders that
overlap with high-risk conditions for severe COVID-19 (15, 51, 52, 68).
Ultimately in vivo clinical data is necessary to test the
hypothesis of whether pan-caspase inhibition can prevent inflammasome
activation in early onset SARS-CoV-2 patients and subsequent lymphopenia
and sequelae development. Furthermore, the pan-caspase inhibitor, EMR
has been shown in a bioinformatics computational screen to bind to the
COVID-19 receptor ACE2, suggesting a potential block to cell entry (69).
In a separate unrelated study, a screen of ~6,070 drugs
with a known 28 previous history of use in humans was conducted to
identify compounds that inhibit the activity of SARS-CoV-2 main protease
Mpro in vitro (70). EMR was shown to be among 50 compounds with
activity against Mpro with an overall hit rate <0.75%.
Preliminary evidence on this multimodal therapeutic effect of EMR raise
a relevant key question that will need to be answered through a
randomized clinical trial in the setting of COVID-19 (Figure 6).
An important aspect of our study is the demonstration of caspase-1
expression well past the acute stage of COVID-19, suggesting a role in
the convalescent phase or disease sequelae. Such persistent changes can
not only be limited to immune system cells but can be seen in tissues
such as endothelial cells, which could be a causal impact on multiple
organ systems (19, 39, 71). Assessing the sequelae, such as fatigue,
dyspnea, cough, joint pain, anosmia, among others (32, 72), in
correlation with the changes in caspase molecules in natural history
studies are warranted. Sequelae targeting populations where caspase
elevations are more common, such as the elderly, and those with other
co-morbid conditions, such as heart disease, diabetes, hypertension,
provides further evidence for the association of caspases with poor
outcomes from COVID-19 (1). Dampening the inflammatory response early in
the disease process may be a strategy to prevent sequelae, such as in
rheumatic fever, where treating streptococcus early on in the disease
through the co-treatment of penicillin and anti-inflammatories can
prevent severe disease sequelae.